The achievement of a robust pathogenic infection of mice with HIV-1 resembling natural HIV-1 infection in people would greatly facilitate understanding of HIV-1 biology in vivo, development of AIDS vaccines, and testing of new therapeutics. Early studies conducted in cell lines indicated that there are major blocks to HIV-1 replication in mouse cells at several points in the viral life cycle, but recent research suggests that HIV-1 infection of primary mouse cells and mice in vivo is in principle possible. This research indicates that the major impediment limiting HIV-1 replication in mouse cells is a defect in the assembly of the viral Gag polyprotein that reduces Gag binding to the plasma membrane and viral particle export. Research has implicated the membrane-binding function of the MA protein within Gag in this defect;however, early multimerization steps are required before Gag is able to bind to membranes at all. The overall goal of this fellowship application is to continue the investigation of a novel defect in cytosolic Gag-Gag interaction in mouse cells that may influence Gag membrane binding, assembly and virion export. Preliminary results using FRET and fractionation techniques indicate that the presence of MA in Gag impedes early Gag multimerization steps in mouse cells. These results support the hypothesis that the murine restriction of HIV- 1 assembly is due to inhibition of Gag-Gag multimerization prior to membrane binding, as a result of interactions between MA and factors present in mouse cells. Identification and mutagenesis of the regions in MA involved in this restriction may improve Gag multimerization in mouse cells, facilitate virion export, and improve HIV-1 replication in mice. This hypothesis will be tested in three Specific Aims: 1) To study Gag-Gag interactions in mouse cells using FRET and functional assays to pinpoint the roles of MA and NC in Gag multimerization;2) To identify the sites of HIV-1 assembly in mouse cells, using staining and cell fusion techniques;and 3) To identify MA mutants with enhanced multimerization by construction and functional testing of MA deletion and alanine scanning mutants, and to test whether such mutants cloned into full-length virus are able to support the full replication cycle of HIV-1. The proposed studies will employ virological and molecular biology methods;flow cytometry, confocal microscopy, FRET and immunofluorescence techniques;and primary macrophage and astrocyte cultures. This research is aimed at understanding and circumventing the block to HIV-1 replication in mouse cells, and may help in the development of a powerful model of HIV-1 infection in mice. Such a model would be an enormous step forward in the search for vaccines and therapeutics for HIV-1 infection.